Regulation Mechanism Of SIRT1 Mediated TGF?1 Signaling Pathway In Liver Fibrosis

[Objective]TGF?/smad pathway is the most common pathway by which transforming growth factor-?(TGF?)plays a key role in the initiation of hepatic fibrosis and the activation and transformation of hepatic stellate cells.Silent information regulator 1(SIRT1)is a reduced coenzyme I-dependent histone deacetylase,which can affect the physiological process of hepatic fibrosis by inhibiting TGF?,but the mechanism of regulation is still not clear.To study SIRT1-mediated regulation of TGF? signaling pathway in hepatic fibrosis can provide a scientific basis for the treatment of hepatic fibrosis.We selected high-fat diet-induced Rhizoctonia sinensis and rat hepatic stellate cells(HSCT6)as subjects to study TGF?/Smad signaling pathway related factors,including Smad7 and phosphorylated Smad3(phospho-Smad3),transforming growth factor-?1(TGF?1),a-smooth muscle actin(a-SMA),and SIRT1's levels.Based on this,we discussed(1)the effect of SIRT1 on TGF?/Smad pathway in the process of liver fibrosis;(2)SIRT1-mediated regulation of TGF? signaling pathway in hepatic fibrosis.[Methods]In the first part,the effect of SIRT on TGF?/Smad pathway factors in the process of liver fibrosis was studied.The liver biopsies were performed on approximately 100 macaques and hepatic fibrosis staging were performed according to Masson trichrome staining.The control group,the mild liver fibrosis group,the moderate liver fibrosis group,and the severe liver fibrosis group were selected from among them and 8 animals in each group.HE staining was used to observe histopathological lesions.The expression level and distribution of protein were detected by immunohistochemistry.The second part,SIRT 1-mediated regulation of TGF?signaling pathway in hepatic fibrosis.SIRT1 agonist(resveratrol)and SIRT1 inhibitor(nicotinamide)were applied to HSCT6,cellular mRNAs and proteins were extracted at 6h,12h and 24h,respectively.The gene expression levels of SIRT1,Smad7,Smad3,TGF?1 and ?-SMA were detected by fluorescent quantitative PCR.The protein expression levels of SIRT1,Smad7,Smad3,TGFp 1 and a-SMA were detected by Western Blot.[Results]Histopathological results showed that a small amount of steatosis and slight fibrosis around the hepatic sinusoids were observed in mild fibrosis group compared with the control group.Perivascular fibrosis and focal inflammatory cell infiltration were observed in the moderate hepatic fibrosis group.Severe liver fibrosis group showed bridging fibrosis,diffuse fatty degeneration,and inflammatory cell infiltration.Blood biochemical results showed that as liver fibrosis increased,AST and GGT were significantly higher(P<0.05).Compared with the control group,ALT levels in each group were significantly higher(P<0.05).The AST/ALT values in the mild liver fibrosis group and the moderate liver fibrosis group were significantly lower than those in the control group(P<0.05),and the AST/ALT values in the severe liver fibrosis group were significantly increased(P<0.05).The results of immunohistochemistry showed that with the increase of liver fibrosis,SIRT1 and Smad7 expression decreased significantly(P<0.01),and the expression of Phospho-3,TGF-?1 and ?-SMA increased significantly(P<0.01).The results of fluorescence quantitative PCR showed that compared with the control group,the expression of SIRT1 was significantly increased in resveratrol-stimulated HSCT6 cells(P<0.05),while the expression of Smad7 increased and the expression of a-SMA decreased,but the difference was not significant.After resveratrol stimulated cells for 12h,SIRT1 expression was significantly increased(P<0.01),TGF-?1 expression was significantly decreased(P<0.01),and a-SMA expression was significantly decreased(P<0.05).After niacinamide stimulated cells for 12h,the expression of TGF-?1 was significantly increased(P<0.05),and the expression of ?-SMA was significantly increased(P<0.01).The expression of SIRT1 was significantly increased(P<0.01),the expression of TGF-?1 and ?-SMA was significantly decreased(P<0.01),and the expression of Smad7 and Smad3 was significantly increased(P<0.01)after resveratrol stimulated cells for 24h.After 24 hours of nicotinamide stimulation,the expression of Smad3 and TGF-?1 were significantly increased(P<0.05),and the expression of a-SMA was significantly increased(P<0.01).Western Blot results showed that after resveratrol-stimulated cells 6 h,12 h,and 24 h,the SIRT1 protein was significantly increased(P<0.01),and TGF-?1 protein expression was significantly decreased(P<0.05),?-SMA protein was significantly decreased(P<0.05),phospho-Smad3 protein was significantly decreased(P<0.05).Smad7 protein had no significant difference at 6 h,and was significant increased(P<0.05)at 12 h and 24 h.After 6h,12h and 24h stimulation of nicotinamide,SIRT1 protein was inhibited and significantly decreased,Smad7 protein was significantly decreased(p<0.05),phospho-Smad3 protein expression was significantly increased(P<0.05),TGF-?1 protein expression.The amount was significantly increased(P<0.05),the expression of a-SMA protein was significantly increased(P<0.05).[Conclusion](1)From healthy,mild liver fibrosis,moderate liver fibrosis to severe liver fibrosis,the degree of inflammation in liver tissue of macaque monkeys gradually increased.The degree of hepatic steatosis gradually deepened and the range gradually expanded.AST and GGT were positively correlated with liver fibrosis severity.The ST/LT ratio was negatively related to the degree of fibrosis in mild and moderate liver fibrosis,and significantly increased in severe liver fibrosis.TG was significantly elevated in severe liver fibrosis The expression of SIRT1 was negatively correlated with TGF-?1,?-SMA and phospho-Smad3 in liver tissue,and positively correlated with the expression of Smad7.